JP4714851B2 - DNA base sequence having gene expression suppression function - Google Patents

Description

The present invention provides, for example, a double-base overlapping sequence and / or TTGGT at a predetermined position relative to the target gene.
And / or a method for suppressing the expression of a target gene by culturing a host having an ACACA base sequence in the presence of retinoic acid.

  Conventionally, it has been known that gene expression is controlled by retinoic acid (Non-Patent Documents 1 to 3).

  For example, in Non-Patent Document 1, when an Ikaros-binding site (GGGAAGGGGAT: SEQ ID NO: 1) present in an intron of a kappa opioid receptor gene is used, gene expression is performed in the presence of retinoic acid. It is disclosed that suppression occurs. The gene expression suppression mechanism is that the expression of Ikaros-1 transcription factor is first induced by retinoic acid, and then the induced Ikaros-1 transcription factor binds to the Ikaros binding site present in the intron described above. Thus, the expression of the kappa opioid receptor gene is suppressed.

  Non-Patent Document 2 discloses that protein expression and promoter activity of a gene called RPE65 are suppressed by retinoic acid. The repression requires the entire 1.3 kb RPE65 promoter, and a specific short base sequence specifically involved in repression has not been identified.

  Similarly, Non-Patent Document 3 discloses that the expression of BMP (Bone Morphogenetic Protein) 4 is suppressed by retinoic acid. Although it has been shown that the BMP4 promoter is involved in the suppression of the expression, it has not been investigated which base sequence of the promoter region plays an important role.

Xinli Hu et al., "The Journal of Biological Chemistry", 2001, 276, p. 4597-4603 Yumei Chen et al., “Molecular Vision”, 2003, Vol. 9, p.345-354 Deborah L. Thompson et al., "Molecular and Cellular Biology", 2003, Vol. 23, p.2277-2286

  An object of this invention is to provide the expression suppression method of a target gene in view of the situation mentioned above.

  As a result of earnest research to solve the above problems, in the host DNA, (a) 5TG upstream of the target gene promoter and / or 3TG downstream of the stop codon of the target gene, (TG) n, (CA ) n, (GT) n and (AC) n (where n is an integer of 3 or more), and / or (b) One or more base sequences of TTGGT and / or ACACA are introduced inside and / or 5 ′ upstream of the target gene promoter and / or 3 ′ downstream of the stop codon of the target gene, a double base overlapping sequence and / or TGTGT and The inventors have found that expression of a target gene can be suppressed by culturing a host having a host DNA into which an ACACA base sequence has been introduced in the presence of retinoic acid, thereby completing the present invention.

  The present invention includes the following.

(1) In the host DNA, (TG) n, (CA) n, (GT) n and (AC) 5a upstream of the target gene promoter and / or 3 ′ downstream of the stop codon of the target gene ) introducing one or more double-base overlapping sequences selected from the group consisting of n, and / or (b) 3 'of the target gene promoter and / or 5' upstream and / or the stop codon of the target gene A first step of introducing one or more base sequences of TTGGT and / or ACACA downstream, and a second step of culturing a host having the host DNA obtained in the first step in the presence of retinoic acid, n is an integer greater than or equal to 3, The expression suppression method of a target gene characterized by the above-mentioned.
(2) The method according to (1), wherein the one or more double-base overlapping sequences are introduced 5 ′ upstream of the target gene promoter and 3 ′ downstream of the stop codon of the target gene.
(3) One or more nucleotide sequences of TTGGT and / or ACACA are introduced into the promoter of the gene of interest and / or upstream of the 5 ′ side and downstream of the codon 3 ′ of the gene of interest (1) The method described.
(4) Any one of (1) to (3), wherein the 5 ′ upstream side of the target gene promoter is the position of the first base to the 2000th base 5 ′ upstream from the target gene promoter The method according to claim 1.
(5) The 3 ′ downstream of the stop codon of the target gene is the position of the 1st to 1000th bases 3 ′ downstream from the stop codon, (1) to (3) Any one of the methods.
(6) The method according to any one of (1) to (5), wherein the host is a mammalian cell.
(7) The method according to any one of (1) to (6), wherein the retinoic acid is present at a concentration of 0.01 to 1000 nM.
(8) The method according to any one of (1) to (7) above, comprising controlling the expression suppression level of the target gene by controlling the expression level of PTB (polypyrimidine tract-binding protein) in the host. .

  According to the present invention, for example, expression of a gene of interest used in gene therapy, production of a transgenic animal, regenerative medicine, and the like can be suppressed.

  Hereinafter, the present invention will be described in detail.

  The method of the present invention (hereinafter referred to as “the present method”) is a method of suppressing the expression of a target gene in host DNA. In this method, first, in the host DNA, (TG) n, (CA) n, (GT) n and (AC) 5 ′ upstream of the target gene promoter and / or 3 ′ downstream of the stop codon of the target gene. ) Introducing one or more double-base overlapping sequences selected from the group consisting of n. Alternatively, in the host DNA, the base sequence of TGTGT and / or ACACA (hereinafter referred to as `` 5 base sequence '') inside the promoter of the target gene and / or upstream 5 ′ and / or 3 ′ downstream of the stop codon of the target gene. ) Is introduced. Here, 5 ′ upstream means the direction following the 5 ′ end of the target gene promoter, while 3 ′ downstream means the direction following the 3 ′ end of the stop codon of the target gene. .

  Next, a host having host DNA into which a 2-base overlapping sequence and / or a 5-base sequence has been introduced is cultured in the presence of retinoic acid. At this time, expression of a transcript or protein encoded by the target gene is suppressed.

  In the present invention, the target gene may be any gene as long as it is a gene already present in the host DNA before application of the present method. It may be a gene introduced into. The target gene promoter means a promoter that controls the target gene. The target gene promoter may exist 5 ′ upstream from the target gene or may exist inside the target gene. The target gene promoter may be any promoter that already exists for the target gene in the host DNA prior to application of this method. For example, the promoter may be an inherently existing promoter, It may be a promoter that has been artificially introduced before. Examples of artificially introduced promoters include SV40 promoter, CMV promoter, HSV-TK promoter, retrovirus promoter, and the like.

  On the other hand, host DNA means DNA having a target gene and a target gene promoter. Examples of host DNA include genomic DNA and mitochondrial DNA in a predetermined organism. Moreover, for example, plasmids, shuttle vectors, helper plasmids, phage DNAs, virus vectors, etc. that already exist in the host before application of this method can also be used as host DNA.

  Hosts include, for example, animal cells including mammalian cells such as HEK293 cells, COS cells and CHO cells, Bacillus genus such as Bacillus subtilis, Escherichia genus such as Escherichia coli and Pseudomonas genus such as Pseudomonas putida. Bacteria belonging to, yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, insect cells such as Sf9, and Gramineae (Oryza sativa, corn (Zea mays)), Brassicaceae ( Examples include plants such as Arabidopsis thaliana, solanaceae (Nicotiana tabacum), and legumes (Glycine max), with mammalian cells being particularly preferred.

  Below, this method in the case of introduce | transducing a 2 base duplication sequence into host DNA is demonstrated.

In the present invention, the double-base overlapping sequence means a sequence in which two bases TG, CA, GT or AC overlap and are juxtaposed. Here, the double-base overlapping sequence is represented as (TG) n, (CA) n, (GT) n or (AC) n. n is an integer of 3 or more. In other words, the two-base overlapping sequence is a sequence in which two bases TG, CA, GT or AC are juxtaposed three times or more. n is preferably an integer of 3 to 100, and particularly preferably an integer of 20 to 40. In this method, the longer the double-base duplication sequence, the more the expression of the target gene can be significantly suppressed. Hereinafter, the double-base overlapping sequence is referred to as “repeat sequence”.

  Hereinafter, this method will be specifically described with reference to FIG. In FIG. 1, the black box located 5 ′ upstream of the promoter is a repeat sequence insertion site 5 ′ upstream of the target gene promoter in the host DNA. On the other hand, a black box 3 'downstream of the target gene is a repeat sequence insertion site 3' downstream of the stop codon of the target gene.

  In this method, first, a DNA fragment containing a repeat sequence is prepared. For example, a DNA fragment containing a repeat sequence can be easily obtained by mixing and annealing two DNA oligomers each corresponding to a single strand of a DNA fragment containing a repeat sequence. In addition, a DNA fragment containing a repeat sequence can be easily amplified as a PCR product by PCR using a DNA fragment containing a repeat sequence as a template and a primer complementary to the nucleotide sequences at both ends.

  Next, as shown in FIG. 1, a DNA fragment containing a repeat sequence is introduced 5 ′ upstream of the target gene promoter and / or 3 ′ downstream of the stop codon of the target gene in the host DNA. In addition, it is preferable to introduce repeat sequences both 5 ′ upstream of the target gene promoter and 3 ′ downstream of the stop codon of the target gene in the host DNA. The position where the repeat sequence should be introduced 5 ′ upstream of the target gene promoter is, for example, the first base to the 2000th base, preferably the first base to the 500th base, particularly preferably 5 ′ upstream from the target gene promoter. Is the position of the first base to the 100th base. On the other hand, the position where the repeat sequence is to be introduced 3 'downstream of the stop codon of the target gene is, for example, the first base to the 1000th base, preferably the first base to 500th, downstream of the stop codon of the target gene. The position of the base, particularly preferably the position of the first base to the 100th base.

  Examples of the method for introducing a DNA fragment containing a repeat sequence into a predetermined position 5 ′ upstream of the target gene promoter of the host DNA and / or 3 ′ downstream of the stop codon of the target gene include, for example, homologous recombination methods. It is done. Here, the DNA fragment containing the repeat sequence may be, for example, a DNA fragment such as a PCR product or a part of the genome, and may be in a form contained in a plasmid, shuttle vector, helper plasmid, phage DNA, viral vector, or the like. . For example, plasmids include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, etc.) YEp system, YCp system such as YCp50), etc., and phage DNA includes λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal virus vectors such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used. Alternatively, by a sleeping beauty that combines a fish-derived transposon (for example, T2 etc.) and a transposase, the repeat sequence is 5 ′ upstream of the target gene promoter of the host DNA and / or 3 ′ downstream of the stop codon of the target gene. A host individual introduced at a predetermined position may be selected.

  When the host is an animal cell such as a mammalian cell, examples of the method for introducing a DNA fragment containing a repeat sequence into the animal cell include an electroporation method, a calcium phosphate method, and a lipofection method.

  When the host is a bacterium, examples of the method for introducing a DNA fragment containing a repeat sequence into the bacterium include a method using calcium ions and an electroporation method.

  When the host is yeast, examples of the method for introducing a DNA fragment containing a repeat sequence into yeast include an electroporation method, a spheroplast method, and a lithium acetate method.

  When the host is an insect cell, examples of the method for introducing a DNA fragment containing a repeat sequence into an insect cell include a calcium phosphate method, a lipofection method, and an electroporation method.

  When the host is a plant, the whole plant, plant organ (e.g. leaf, petal, stem, root, seed, etc.), plant tissue (e.g. epidermis, phloem, soft tissue, xylem, vascular bundle, etc.) or plant culture Cells are used. Examples of a method for introducing a DNA fragment containing a repeat sequence into a plant include an electroporation method, an Agrobacterium method, a particle gun method, and a PEG method.

  As described above, a DNA fragment containing a repeat sequence can be introduced into a host.

  Whether a repeat sequence has been incorporated at a predetermined position 5 ′ upstream of the target gene promoter of the host DNA and / or 3 ′ downstream of the stop codon of the target gene is determined by PCR, Southern hybridization, and Northern. It can be performed by a hybridization method or the like. For example, DNA is prepared from a transformant, and PCR is performed by designing a primer specific to a region that can be amplified when a repeat sequence is incorporated at a predetermined position of host DNA. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, Confirm that the repeat sequence has been integrated into the host DNA. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or enzyme reaction may be employed.

  Next, in this method, a host (transformant) having host DNA having a repeat sequence is cultured in the presence of retinoic acid. The culture conditions are as follows, for example, when the host is a mammalian cell such as HEK293 cells; temperature: 30 to 38 ° C., preferably 36 to 37 ° C., pH: pH 7.0 to 8.0, preferably PH is 7.4 to 7.6, culture time: 10 to 80 hours, preferably 20 to 30 hours.

Any type of retinoic acid may be used, and examples thereof include all-trans retinoic acid, 13-cis-retinoic acid, and 9-cis-retinoic acid. Further, retinol, retinal, retinyl acetate, retinyl palmitate and the like may be used. The amount of retinoic acid added at the time of culture is, for example, a concentration of 0.01 to 1000 nM, preferably 1 to 1000 nM, particularly preferably 100 to 1000 nM with respect to the medium.
Furthermore, PTB (polypyrimidine tract-binding protein) is involved in the suppression of expression by retinoic acid of the present invention, and the degree of expression suppression by retinoic acid can be controlled by controlling the amount of PTB expression in the host.
For example, when PTB is cotransfected into a cultured cell HEK293 together with a reporter vector to encode PTB and the PTB is overexpressed, gene expression suppression by a retinoic acid suppression sequence (26mer) is promoted (see Example 7 below). . Conversely, co-transfecting an siRNA vector against PTB to cause RNA interference and reduce the function of PTB inhibits expression suppression by retinoic acid (see Example 8 below).
That is, by combining a PTB expression vector with a retinoic acid inhibitory sequence, control of gene expression by retinoic acid can be further strengthened or weakened.

  According to the method described above, the expression of the target gene in the host can be significantly suppressed. Confirmation that the expression of the target gene could be suppressed by this method can be performed at the gene level such as transcription and the protein level, for example. For example, confirmation at the gene level such as transcription includes PCR, Southern hybridization, Northern hybridization, and the like. Examples of confirmation at the protein level include Western blotting using an antibody against a protein encoded by the target gene, flow cytometry, and the like. Alternatively, when the protein encoded by the target gene is an enzyme, suppression of expression of the target gene can be confirmed by measuring the enzyme activity. If the expression of the target gene in the host to which this method is applied is significantly reduced at the gene level or protein level compared to the expression of the target gene in the host to which this method is not applied, It can be judged that gene expression suppression is good.

  On the other hand, according to this method for introducing a repeat sequence into the host DNA described above, inside the host gene promoter and / or 5 ′ upstream and / or 3 ′ downstream of the stop codon of the target gene in the host DNA Furthermore, expression of the target gene can be suppressed by introducing one or more 5-base sequences and culturing a host having host DNA into which the 5-base sequences have been introduced in the presence of retinoic acid. In this method, one or a plurality (for example, 4 or more) of 5 base sequences are introduced into the target gene promoter and / or upstream 5 ′. In addition, one or a plurality (for example, 4 or more) of 5 base sequences is introduced downstream of the target gene at the 3 ′ side of the stop codon.

  In this method, introduction of a repeat sequence in host DNA and introduction of a 5-base sequence may be combined.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

  Moreover, the following Examples 1-4 are demonstrated according to FIG. In FIG. 2, the black box located 5 ′ upstream of the SV40 promoter in the pGL3-promoter vector is the repeat sequence insertion site. The black box located 3 ′ downstream of the poly (A) signal is an enhancer site of pGL3-promoter vector (Promega) and a repeat sequence insertion site. Note that the enhancer of the pGL3-promoter vector used in this example has been removed.

[Example 1] Insertion of 8 times TG ((TG) ₈ ) repeat sequence 3 ′ downstream of target gene (luciferase gene) 25 μM each of the following 2 types of DNA oligomers (SEQ ID NOs: 2 and 3) And heated at 95 ° C. for 15 minutes and then left at 55 ° C. for 15 minutes.
for: 5'-CGCGGATCCTGTGTGTGTGTGTGTGGGATCCGCG-3 '(SEQ ID NO: 2)
rev: 5'-CGCGGATCCCACACACACACACACAGGATCCGCG-3 '(SEQ ID NO: 3)

  After returning to room temperature, the annealed DNA fragment was cleaved at the 5 'and 3' end bases with Bam HI, and the resulting short fragment was removed by a spin column to obtain a DNA fragment (8 times TG repeat sequence). Including).

  On the other hand, pGL3-promoter vector (Promega) was treated with alkaline phosphatase after cleaving the enhancer site with Bam HI.

  Subsequently, the DNA fragment containing the 8-times TG repeat sequence obtained as described above was introduced into the enhancer site of the pGL3-promoter vector after the alkaline phosphatase treatment and ligated. The obtained ligation product was used to transform E. coli, and colonies containing the target plasmid were selected. Escherichia coli was cultured from the selected colonies, lysed by the alkaline method, and then the ligation product was isolated by using a Qiagen DNA purification kit. As shown in FIG. 2, the resulting ligation product is an enhancer site present 3 ′ downstream of the poly (A) signal of the pGL3-promoter vector, ie, 270 bases downstream from the stop codon of the luciferase gene. , 8 times TG repeat sequence is inserted.

Subsequently, HEK293 cells (2 × 10 ⁵ ) were seeded in a 34 mm diameter well of a cell culture plate, and the ligation product was transiently transfected using a gene therapy system. Twenty-four hours after transfection, retinoic acid was added to the culture to a concentration of 1 μM, and the culture was further cultured for 24 hours.

  After culturing, the cells were collected and luciferase activity was measured. In order to measure the efficiency of transfection, the pRL-CMV vector was also transfected at the same time to calibrate the luciferase activity. The pRL-CMV vector is an internal control reporter vector that contains the Renilla luciferase gene under the control of the CMV promoter and stably expresses the luciferase. As a control, the transfected cells were cultured in the same manner in the absence of retinoic acid.

  The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 3, when 1 μM all-trans retinoic acid was added, the luciferase activity in the cells reached 75.0% compared to the luciferase activity (100%) in the absence of retinoic acid (0 μM). Suppressed.

[Example 2] Insertion of 26 CA ((CA) 26) repeat sequence 3 'downstream of luciferase gene In this example, enhancer site of pGL3-promoter vector (270' 3 'from the codon of luciferase gene) The case where the CA repeat sequence was inserted 26 times downstream of the base was examined.

  Except that the DNA oligomers shown below (SEQ ID NOs: 4 and 5) were used to produce annealing products (including 26 times CA repeat sequence) and that they were branded by KOD before ligation, as in Example 1. E. coli transformation, colony selection, ligation product isolation, HEK293 cell transfection, cell culture, luciferase measurement, and the like were performed.

5'-CACACACACACACACACACACACACACACACACACACACACACACACACACA-3 '(SEQ ID NO: 4) (5' phosphorylated)
5'-TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG-3 '(SEQ ID NO: 5) (5' phosphorylated)

  The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 4, when 1 μM all-trans retinoic acid was added, the luciferase activity in the cells was up to 64.0% compared to the luciferase activity (100%) in the absence of retinoic acid (0 μM). Suppressed.

[Example 3] Insertion of 26 times TG ((TG) ₂₆ ) repeat sequence 5 'upstream of SV40 promoter (target gene promoter) In this example, 5 of SV40 promoter of pGL3-promoter vector shown in Fig. 2 The case where 26 TG repeat sequences were inserted upstream of the 17 bases on the side was examined.

The two DNA oligomers (SEQ ID NOs: 4 and 5) shown in Example 2 were mixed so as to be 25 μM each, heated at 95 ° C. for 15 minutes, and then allowed to stand at 55 ° C. for 15 minutes.
After returning to room temperature, a DNA fragment (containing 26 times TG repeat sequence) was obtained.

  On the other hand, pGL3-promoter vector (Promega) was treated with alkaline phosphatase after cutting 5 ′ upstream of the SV40 promoter with Xho I. Subsequently, the DNA fragment containing the 26 times TG repeat sequence obtained as described above was introduced upstream of the SV40 promoter 5 ′ side 17 bases of the alkaline phosphatase-treated pGL3-promotervector, branded by KOD, and then ligated. . Using the obtained ligation product, transformation of E. coli, selection of colonies, isolation of the ligation product, transfection into HEK293 cells, cell culture, measurement of luciferase and the like were performed in the same manner as in Example 1. .

  The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 5, when 1 μM all-trans retinoic acid was added, the luciferase activity in the cells was up to 87.9% compared to the luciferase activity (100%) in the absence of retinoic acid (0 μM). Suppressed.

[Example 4] Insertion of 26 times TG ((TG) ₂₆ ) repeat sequence 5 'upstream of SV40 promoter and 26 times of CA ((CA) ₂₆ ) repeat sequence 3' downstream of luciferase gene In this example, the TG repeat sequence was inserted 26 times upstream of the SV40 promoter 5 ′ side 17 bases upstream of the pGL3-promoter vector shown in FIG. The case where 26 times CA repeat sequence was inserted was examined.

  Each repeat sequence was inserted into the pGL3-promoter vector in the same manner as described in Examples 2 and 3.

  Subsequently, using the obtained ligation product, transformation of E. coli, selection of colonies, isolation of the ligation product and transfection into HEK293 cells were performed in the same manner as in Example 1.

Twenty-four hours after transfection, retinoic acid was added to the cultures at concentrations of 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM, respectively, and further cultured for 24 hours.
After culturing, the cells were collected and luciferase activity was measured.

  The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 6, when all-trans retinoic acid was added, the luciferase activity in the cells was suppressed in a concentration-dependent manner compared to the luciferase activity in the absence of retinoic acid (0 nM) (100%). When 100 nM all-trans retinoic acid was added, luciferase activity was suppressed to 28.7%.

  As shown in the above examples, the longer gene compared to the short repeat sequence has a higher effect of suppressing the expression of the target gene in the presence of retinoic acid, and the 5 ′ upstream of the target gene promoter and 3 of the target gene. It was found that the introduction of a repeat sequence on the downstream side had a higher effect of suppressing the expression of the target gene in the presence of retinoic acid than that introduced into only one.

[Example 5] Insertion of a target gene (luciferase gene) promoter and a 5 base sequence 3 'downstream of the stop codon of the target gene In this example, the promoter of the pGL3-basic vector (Promega) shown in FIG. The case where the promoter sequence (SEQ ID NO: 6) of the Xenopus slug beta gene was inserted into the site and the intron fragment (SEQ ID NO: 7) of the Xenopus slug beta gene was inserted into the enhancer site was examined. In FIG. 7, the black box located 5 'upstream (57 bases upstream from the start codon) of the luciferase gene in the pGL3-basic vector is the promoter region of the pGL-basic promoter, that is, the promoter sequence of the Xenopus slug beta gene ( SEQ ID NO: 6) Insertion site. In addition, a black box located 3 ′ downstream of the poly (A) signal (270 bases downstream from the stop codon of the luciferase gene) is an enhancer site, that is, an intron fragment (SEQ ID NO: 7) insertion site of the Xenopus slug beta gene. is there. In addition, the commercially available pGL3-basic vector used in this example originally does not contain a promoter sequence and an enhancer sequence.
Moreover, as shown in FIG. 8, the promoter sequence of the Xenopus slug beta gene contains two 5-base sequences, while the intron fragment of the Xenopus slug beta gene contains four 5-base sequences (in FIG. And the underlined base sequence is a 5-base sequence).

  First, Xenopus laevis genomic DNA was used as a template, and 94 ° C .: 15 seconds (denaturation), 55 ° C .: 10 seconds (annealing) and 60 ° C. using the following primer sets (SEQ ID NOs: 8 and 9). : The promoter sequence (SEQ ID NO: 6) of the Xenopus slug beta gene was amplified by PCR in 50 cycles of 10 minutes (extension).

5'-GCACGCGTTAAACTTCACTTGAACATGTTCTAACAG-3 '(SEQ ID NO: 8)
5'-GCCTCGAGTTTCAGTGAGGGAGGGGGACCCCAAGCC-3 '(SEQ ID NO: 9)

  The amplified PCR product was cleaved at both ends using Mlu I and Xho I, and subjected to electrophoresis, thereby purifying the DNA fragment.

  On the other hand, pGL3-basic vector (Promega) was similarly cleaved with Mlu I and Xho I and similarly purified by electrophoresis.

  Subsequently, the DNA fragment containing the promoter sequence (SEQ ID NO: 6) of the above-mentioned Xenopus slug beta gene was ligated to pGL3-basic vector (Promega) from which the promoter site was cleaved.

  Similarly, an intron fragment (SEQ ID NO: 7) of the Xenopus slug beta gene was amplified by PCR using the Xenopus laevis genomic DNA as a template and the following primer sets (SEQ ID NOs: 10 and 11).

5'-GCGGATCCCCGATCGCATGTAGAGATGCAACATTTAC-3 '(SEQ ID NO: 10)
5'-GCGGATCCGGGTTATAGGCTGAATATATTGGGCC-3 '(SEQ ID NO: 11)

The amplified PCR product was cleaved at both ends with Bam HI and subjected to electrophoresis, thereby purifying the DNA fragment.

  On the other hand, the pGL3-basic vector (Promega) containing the promoter sequence of the Xenopus slug beta gene obtained as described above was similarly purified by cleaving the enhancer site with Bam HI and subjected to electrophoresis, followed by alkaline phosphatase treatment. went.

  Next, a DNA fragment containing an intron fragment (SEQ ID NO: 7) of the Xenopus slug beta gene was ligated to pGL3-basic vector (Promega) from which the enhancer site was cleaved.

  Except that the obtained ligation product was used and retinoic acid was added to the culture so as to have a concentration of 10 nM, in the same manner as in Example 1, transformation of E. coli, selection of colonies, isolation of the ligation product, HEK293 Transfection into cells, cell culture, measurement of luciferase, and the like were performed.

The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 9, when all-trans retinoic acid is added, the luciferase activity in the cells is 10 nM all-trans compared to the luciferase activity (100%) in the absence of retinoic acid (0 nM). When retinoic acid was added, luciferase activity was suppressed to 42%.

[Example 6] Insertion of target gene (luciferase gene) promoter and 5 base sequence 3 'downstream of stop codon of target gene In this example, promoter of pGL3-basic vector (Promega) shown in Fig. 7 The case where the human slug (SNAI2) gene promoter sequence (SEQ ID NO: 12) was inserted into the site and the human slug gene intron fragment (SEQ ID NO: 13) was inserted into the enhancer site was examined. In Fig. 7, the black box located 5 'upstream (57 bases upstream from the start codon) of the luciferase gene in the pGL3-basic vector is the promoter region of the pGL3-basic promoter, that is, the promoter sequence of the human slug gene (sequence) Number 12) Insertion site. The black box located 3 'downstream of the poly (A) signal (270 bases downstream from the stop codon of the luciferase gene) is the enhancer site, that is, the insertion site of the intron fragment (SEQ ID NO: 13) of the human slug gene. . In addition, the commercially available pGL3-basic vector used in this example originally does not contain a promoter sequence and an enhancer sequence. In addition, as shown in FIG. 10, the promoter sequence of the human slug gene contains eight 5-base sequences, while the intron fragment of the human slug gene contains five 5-base sequences (in FIG. 10, bold and underlined characters). The base sequence indicated by is a 5 base sequence).
First, using human genomic DNA as a template, 94 ° C: 15 seconds (denaturation), 55 ° C: 10 seconds (annealing), and 60 ° C: 10 minutes (extension) using the following primer sets (SEQ ID NOs: 14 and 15) ), The human slug gene promoter sequence (SEQ ID NO: 12) was amplified by PCR.
5'-GCACGCGTGTGTCTGAGCAGAGCACCTGTTTCG-3 '(SEQ ID NO: 14)
5'-CGCTCGAGCACCCGGCTCCTTTACGAACTGAGCC-3 '(SEQ ID NO: 15)
The amplified PCR product was cleaved at both ends using Mlu I and Xho I, and subjected to electrophoresis, thereby purifying the DNA fragment.
On the other hand, pGL3-basic vector (Promega) was similarly cleaved with Mlu I and Xho I and similarly purified by electrophoresis.
Next, a DNA fragment containing the above-described human slug gene promoter sequence (SEQ ID NO: 12) was ligated to pGL3-basic vector (Promega) from which the promoter site had been cleaved.
Similarly, an intron fragment (SEQ ID NO: 13) of the human slug gene was amplified by PCR using human genomic DNA as a template and the following primer sets (SEQ ID NOs: 16 and 17).
5'-GCGGATCCGTAAAAAGAGAAAAATATATCTAGAAC-3 '(SEQ ID NO: 16)
5'-GCGGATCCTGGGAAAGAAAAGGGAGGGAGAGAAG-3 '(SEQ ID NO: 17)
The amplified PCR product was cleaved at both ends with Bam HI and subjected to electrophoresis, thereby purifying the DNA fragment.
On the other hand, the pGL3-basic vector (Promega) containing the human slug gene promoter sequence obtained above was similarly purified by cleaving the enhancer site with Bam HI and subjected to electrophoresis, followed by alkaline phosphatase treatment. It was.
Next, a DNA fragment containing an intron fragment (SEQ ID NO: 13) of the human slug gene was ligated to pGL3-basic vector (Promega) from which the enhancer site was cleaved.
Except that the obtained ligation product was used and retinoic acid was added to the culture so as to be 2 μM, in the same manner as in Example 1, E. coli transformation, colony selection, ligation product isolation, HEK293 cells were transfected, cell culture, luciferase measurement, and the like were performed.
The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 11, when all-trans retinoic acid was added, the luciferase activity in the cells was 2 μM total compared to the luciferase activity (100%) in the absence of retinoic acid (0 μM). When trans retinoic acid was added, luciferase activity was suppressed to 43%.

[Example 7] Overexpression of PTB (polypyrimidine tract-binding protein) Was examined.
Using human genomic DNA as a template, using the following primer sets (SEQ ID NOs: 18 and 19), 94 ° C: 15 seconds (denaturation), 55 ° C: 10 seconds (annealing), and 60 ° C: 10 minutes (extension) At 40 cycles, the human PTB gene sequence (FIG. 12, SEQ ID NO: 20) was amplified by PCR.
5'-GCGAATTCATGGACGGAATCGTCACTGAAGTTGC-3 '(SEQ ID NO: 18)
5'-GCGTCGACTTAAATTGTTGACTTGGAGAAAGACAC-3 '(SEQ ID NO: 19)
The amplified PCR product was cleaved at both ends using EcoR I and Sal I, and subjected to electrophoresis, thereby purifying the DNA fragment.
On the other hand, pCI vector (Promega) was similarly cleaved with EcoR I and Sal I and similarly purified by electrophoresis.
Next, a DNA fragment containing the above-mentioned human PTB2 gene sequence (SEQ ID NO: 20) was ligated to pCI vector (Promega) from which the multicloning site was cleaved.
The obtained ligation product was co-transfected into HEK293 cells, and retinoic acid was added to the culture so as to have a concentration of 2 μM. Ligation product isolation, transfection into HEK293 cells, cell culture, luciferase measurement, and the like were performed.
The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 13, when all-trans retinoic acid was added, the luciferase activity in the cells was 2 μM total compared to the luciferase activity (100%) in the absence of retinoic acid (0 μM). When trans retinoic acid is added, the luciferase activity is suppressed to 13%, which is significantly lower than the luciferase activity (34%) when the PTB2 expression vector is not co-transfected.

[Example 8] Decreased function of PTB (polypyrimidine tract-binding protein) The case where the target sequence was inserted was examined.
Using the DNA oligomers shown below (SEQ ID NOs: 21 and 22), an annealing product (including 21 base sequences on the 3 ′ side and 34 bases downstream from the stop codon of the PTB1 gene) was prepared.
Five'-
GATCCAACTTCCATCATTCCAGAGAACTGTGAAGCCACAGATGGGTTCTCTGGAATGATGGAAGTTTTTTTTAT-3 '(SEQ ID NO: 21)
Five'-
CGATAAAAAAAACTTCCATCATTCCAGAGAACCCATCTGTGGCTTCACAGTTCTCTGGAATGATGGAAGTTG-3 '(SEQ ID NO: 22)
On the other hand, pSINsi-hH1 DNA (Takara-bio) was cleaved with BamH I and Cla I and purified by electrophoresis.
Next, the annealed product containing the target sequence for the human PTB1 gene described above was ligated to pSINsi-hH1 DNA (Takara-bio) from which the cloning site had been cleaved.
In the same manner as described above, the following DNA oligomer (SEQ ID NOs: 23 and 24) is used to prepare an annealing product (including 21 base sequences 3 bases downstream from the stop codon of the PTB2 gene) and cleave the cloning site. PSINsi-hH1 DNA (Takara-bio) was ligated.
Five'-
GATCCATTGTTCAATGTCATCACCTACTGTGAAGCCACAGATGGGTAGGTGATGACATTGAACAATTTTTTTAT-3 '(SEQ ID NO: 23)
Five'-
CGATAAAAAAATTGTTCAATGTCATCACCTACCCATCTGTGGCTTCACAGTAGGTGATGACATTGAACAATG -3 '(SEQ ID NO: 24)
In the same manner as in Example 4, except that the two ligation products obtained were cotransfected into HEK293 cells and retinoic acid was added to the culture to 2 μM. Selection, ligation product isolation, transfection into HEK293 cells, cell culture, luciferase measurement, and the like.
The measurement results of luciferase activity are shown in FIG. As can be seen from FIG. 14, when all-trans retinoic acid was added, the luciferase activity in the cells was 2 μM total compared to the luciferase activity (100%) in the absence of retinoic acid (0 μM). When trans-retinoic acid is added, the luciferase activity is suppressed only to 98%, which is significantly higher than the luciferase activity when RNA interference with PTB is not performed.

FIG. 1 shows the positional relationship between a target gene and a repeat sequence insertion site. FIG. 2 shows a repeat sequence insertion site in the pGL3-promoter vector in Examples 1 to 4. FIG. 3 shows the measurement results of luciferase activity in Example 1. FIG. 4 shows the measurement results of luciferase activity in Example 2. FIG. 5 shows the measurement results of luciferase activity in Example 3. FIG. 6 shows the measurement results of luciferase activity in Example 4. FIG. 7 shows a 5-base sequence insertion site in the pGL3-basic vector in Example 5. FIG. 8 shows the site of the 5 base sequence in the promoter sequence and intron fragment of the Xenopus slug beta gene. FIG. 9 shows the measurement results of luciferase activity in Example 5. FIG. 10 shows the site of the 5-base sequence in the promoter sequence and intron fragment of the human slug gene. FIG. 11 shows the measurement results of luciferase activity in Example 6. FIG. 12 shows the base sequence of the human PTBB2 gene. FIG. 13 shows the measurement results of luciferase activity in Example 7. FIG. 14 shows the measurement results of luciferase activity in Example 8.

SEQ ID NOs: 2 to 5 are DNA oligomers.
Sequence number 8-11 is a primer.

Claims (6)

Whether one or more double base sequences consisting of (TG) n or (CA) n are introduced into the host DNA 5 ′ upstream of the target gene promoter and 3 ′ downstream of the stop codon of the target gene. Alternatively , a host having the host DNA obtained in the first step and the first step of introducing one double-base overlapping sequence consisting of (TG) n or (CA) n downstream of the stop codon of the target gene. And the second step of culturing in the presence of retinoic acid, wherein n is an integer of 20 to 40 .   2. The method according to claim 1, wherein the 5 ′ upstream side of the target gene promoter is located at the 1st to 2000th bases 5 ′ upstream from the target gene promoter.   2. The method according to claim 1, wherein the 3 ′ downstream side of the stop codon of the target gene is the position of the 1st base to the 1000th base 3 ′ downstream from the stop codon.   The method according to claim 1, wherein the host is a mammalian cell.   The method according to any one of claims 1 to 4, characterized in that the retinoic acid is present in a concentration of 0.01 to 1000 nM.   The method according to any one of claims 1 to 5, comprising controlling the expression suppression level of the target gene by controlling the expression level of PTB (polypyrimidine tract-binding protein) in the host.

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